Abstract

Tumor progression is driven by genetic mutations, but little is known about the environmental conditions that select for these mutations. Studying the transcriptomes of paired colorectal cancer cell lines that differed only in the mutational status of their KRAS or BRAF genes, we found that GLUT1, encoding glucose transporter-1, was one of three genes consistently up-regulated in cells with KRAS or BRAF mutations. The mutant cells exhibited enhanced glucose uptake and glycolysis and survived in low-glucose conditions, phenotypes that all required GLUT1 expression. In contrast, when cells with wild-type KRAS alleles were subjected to a low-glucose environment, very few cells survived. Most surviving cells expressed high levels of GLUT1, and 4% of these survivors had acquired KRAS mutations not present in their parents. The glycolysis inhibitor 3-bromopyruvate preferentially suppressed the growth of cells with KRAS or BRAF mutations. Together, these data suggest that glucose deprivation can drive the acquisition of KRAS pathway mutations in human tumors.

Expression of GLUT1 in matched pairs of isogenic clones. (A) Expression levels of GLUT1 transcripts were determined by real-time PCR and normalized to those of β-actin. Each panel includes the parental line (Parent), which harbors both mutant and wild-type alleles of KRAS or BRAF, two independent clones with only mutant alleles (MUT1 and MUT2) and two independent clones with only wild-type alleles (WT1 and WT2). The data represent the mean and SD of triplicate experiments. The differences between MUT and WT clones were statistically significant in all cases (P < 0.05, Student’s t-test). (B) Expression of GLUT1 membrane-associated protein levels as determined by immunoblotting. Na+,K+-ATPase, a membrane associated protein, was used as a loading control.

Glucose uptake and lactate production in cells with KRAS or BRAF mutations. (A) Glucose uptake, as determined using [3H] 2-deoxyglucose, was normalized to total protein. Differences between MUT and WT clones were statistically significant (P < 0.01, Student’s t-test). (B) Lactate production was normalized to cell number. The differences between MUT and WT clones were statistically significant in all cases (P < 0.03, Student’s t-test). The data represent the mean and SD of triplicate experiments.

KRAS and BRAF mutations confer a selective growth advantage in hypoglycemic conditions. (A) Cells were subjected to a low glucose environment (0.5 mM) for two (RKO and VACO432) or four (HCT116 and DLD1) days, then dissociated and plated in media containing standard concentrations of glucose (25 mM). Colony counts were normalized to those obtained in cells subjected to the same experimental procedure with the exception that standard glucose levels were substituted for low glucose. See (7) for details. The differences between MUT and WT clones were statistically significant in all cases (P < 0.004, Student’s t-test). (B) MUT and WT clones were mixed at the indicated ratios and grown in media with 0.5 mM glucose for two days (RKO) or five days (DLD1). The media was replaced with one containing 25 mM glucose and the cells incubated for another 10–16 days. RNA was purified from the cells that survived and the KRAS or BRAF genes were PCR-amplified and sequenced. G and A nucleotides at the underlined positions in the sequencing chromatograms represent wt and mutant alleles of KRAS, respectively, in DLD1 cells. T and A nucleotides represent wt and mutant alleles of BRAF, respectively, in RKO cells. (C) DLD1 cells in which the mutant KRAS allele had been deleted by targeted recombination (KRAS (−/+)), were plated in low glucose (0.5 mM). After 25–30 days, the few clones that survived were grown in standard glucose (25 mM) and assessed for GLUT1 expression and the sequence of the KRAS gene. Clones which harbored mutant alleles of KRAS (G12D, G13D, or G13C) are indicated, as are clones in which KRAS remained WT. As controls, the same cells (KRAS (−/+)) were plated at limiting dilution in media containing 25 mM glucose and individual clones assessed for GLUT1 expression (“Control Clones”). The parental cells used for these experiments (DLD1, WT) are also included, as were their isogenic counterparts in which the wt rather than the mutant allele was disrupted by homologous recombination (DLD1, MUT). All clones had been growing in media containing 25 mM glucose for at least 20 days when harvested for the assessment of GLUT1 expression by immunoblotting. Na+,K+-ATPase was used as a loading control. A diagram of the selection scheme is provided in fig. S10 and detailed methods are provided in (7).

The glycolysis inhibitor 3-BrPA is selectively toxic to cells with mutant KRAS or BRAF alleles. (A) Colony formation was assessed after 3-BrPA treatment (110 μM) for three days. Colony counts were normalized to those obtained from cells subjected to the same procedure without exposure to 3-BrPA. The differences between MUT and WT clones were statistically significant in all cases (P < 0.008, Student’s t-test). (B) Mice with subcutaneous tumors established from HCT116 (KRAS: G13D/+) or VACO432 (BRAF: V600E/+) cells were injected intraperitoneally with 3-BrPA or phosphate buffered saline (PBS) daily for two weeks. “n” represents the number of mice used in each group. Points and error bars represent the means and SD for each group of mice. Asterisks denote times when there were significant differences between the tumor sizes in the PBS vs. 3-BrPA groups (P < 0.05, Student’s t-test).